I wish to amplify 3.5kb cDNA of a gene of interest and then clone into pjET vector for sequencing. I am having issues with the primer design. I intend to design forward primer at every 500 bp and use the whole mixture for the normal PCR. Suggestions would be appreciated.
Huiquan Zheng
It is not an ideal strategy because you had to demenstrate that these fragment located on the same sequence. I suggest to use the long pcr method. Good luck.
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Avijit Pramanik
it is better to use long range PCR and get the 3.5 kb amplified in one go. for sequencing you can do primer walking from both sides at 500 bp interval.
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